Heat Shock Proteins And Juvenile Chronic Arthritis

The involvement of hsps and immune reactivity to hsps in JCA is apparent from a number of observations.

A. Synovial Membranes

Initially expression of hsp 60 was studied in synovial membranes of rats with adjuvant arthritis and in adult patients with either reumatoid arthritis or osteoarthritis (11). Later on hsp 60 expression was also studied in patients with JCA (12). The monoclonal antibodies used in the first studies were raised in mice: ML-30 against hsp 60 of Mycobacterium leprae,TB-78 against hsp 60 of M. tuberculosis, and F-8 raised against the arthritogenic nonapeptide of M. tuberculosis (Table 2).

For all antibodies used, a similar staining pattern was found; that is, predominant staining of synovial lining ells, blood vessels, and macrophages. There was no correlation between the intensity of staining with the above-mentioned antibodies and the degree of lining cell proliferation or the density or location of lymphoid infiltration. In a second study, a monoclonal antibody to human hsp 60 was developed (LK-1) with a unique specificity for human hsp 60 and not reactive with the bacterial counterpart (12). Another antibody, LK-2, recognizes both the human and the bacterial hsp 60 (Table 2). With the availability of LK-1 and LK-2, the question whether epitopes of human hsp 60 or bacterial hsp 60 are expressed in synovial membranes of patients with JCA could be addressed. Expression of human hsp 60 (LK1+) was found in the cytoplasm of

Juvenile Chronic Arthritis and Hsp Table 2 Expression of Hsp 60 in Synovial Membranes of Rats and Humans

Monoclonal antibody Ig class

Reactivity to epitope within aminoacid Reference

Staining pattern

ML-30

IgGl 280-303 of mycobacterial 13 hsp 60

Cytoplasmic staining ol synovial lining and endothelial cells As with ML-30

TB-78

IgGl 170-234 of mycobacterial hsp 60

IgM 180-188 of mycobacterial hsp 60

IgGl 383-447 of human hsp 60

IgG 1 383-419 of human and bacterial hsp 60

11,15 As with ML-30

12 12

As with ML-30 As with ML-30

lining cells and endothelial cells and macrophages, all with a high level of expression of HLA-DR (11,16). The pattern of expression of human hsp 60 in synovial membranes of patients with JCA as detected by LK-1 parallelled exactly the staining pattern of the antibodies, ML-30, TB-78, and F-8 (unpublished observations by the authors). The cross-reactive LK-2 antibody also showed a similar staining pattern. With exception of F-8, it seems likely that cross reactivity with human hsp 60 was the basis for this result. The monoclonal antibody F-8 recognizes the 180-188 amino acid sequence of mycobacterial hsp 60, a sequence not present in human hsp 60. Overall it can be concluded that both endogenous hsp 60 and microbial hsp 60 may be expressed in synovial tissue of patients with JCA and that hsp 60 is localized mainly in synovial lining cells, endothelial cells of blood vessels, and macrophages.

B. Cell Surface Expression of Heat Shock Protein

Whether or not (processed) hsp 60 fragments are expressed on the cell surface, for example, of synovial lining cells or macrophages is a controversial issue due in part to the lack of suitable reagents. Class I-restricted CD8+ T cells, raised against the bacterial hsp 60, recognize macrophages that were subjected to various stress stimuli, including interferon gamma activation and viral infection (17). The proliferative response of y8 T cells to the Daudi B-cell line can be inhibited by a rabbit anti-hsp 60 antiserum (18). Ascites of ML-30, a monoclonal antibody specific for an epitope encoded by amino acids 280-303 of mycobacterial hsp 60 (13), shows surface staining of murine bone marrow-derived macrophages (19). However, purified monoclonal antibody or tissue culture derived ML-30 did neither show surface staining of macrophages nor a variety of murine and human myelomonocytic and B-cell lines (20,21). Our own observations (Fig. 1) indicate that on heat shock treatment, peripheral blood monocytes of normal individuals as well as patients with JCA express human hsp 60 determinants as evidenced by increased cell surface staining with LK-1. These results suggest that epitopes of human hsp 60 are present in inflamed synovial tissue of patients with JCA and have, when properly expressed, the potential to initiate a humoral and/or cellular immune response.

C. Juvenile Chronic Arthritis a T-Cell-Mediated Disease?

Even without knowledge of the nature and distribution of potential autoantigens that may be involved, there are various clinical arguments to consider JCA a T-cell-mediated autoimmune disease. The first argument in support of this is

10° 101 102 103 104 10° 101 102 103 104 fluorescence intensity fluorescence intensity

Figure 1 Induction of cell surface hsp 60 expression on human monocytes by heat shock treatment. Human peripheral blood mononuclear cells were incubated for 30 min at either 37°C (heavy tracing) or 41 °C (light tracing), followed by 2 hr at room temperature. Cells were stained with biotinylated LK-1 (A), phycoerythrin conjugated anti-HLA-DR (B), or biotinylated R73 OX6, OX18, or OX19 (anti-rat leukocyte antigens; panel D). In a second step, cells were incubated with streptavidin-phycoerythrin (A and D) and fluoresceinated CD14 (pan-monocyte). Cells were analyzed on a FACStar Plus flowcytometer (Becton Dickinson); shown are histograms of CD14+ gated monocytes. Note that apart from LK-1 (panel A), heat shock treatment does not change expression of HLA-DR (B) or CD14 (C), nor does it lead to binding of irrelevant MAbs (D).

10° 101 102 103 104 10° 101 102 103 104 fluorescence intensity fluorescence intensity

Figure 1 Induction of cell surface hsp 60 expression on human monocytes by heat shock treatment. Human peripheral blood mononuclear cells were incubated for 30 min at either 37°C (heavy tracing) or 41 °C (light tracing), followed by 2 hr at room temperature. Cells were stained with biotinylated LK-1 (A), phycoerythrin conjugated anti-HLA-DR (B), or biotinylated R73 OX6, OX18, or OX19 (anti-rat leukocyte antigens; panel D). In a second step, cells were incubated with streptavidin-phycoerythrin (A and D) and fluoresceinated CD14 (pan-monocyte). Cells were analyzed on a FACStar Plus flowcytometer (Becton Dickinson); shown are histograms of CD14+ gated monocytes. Note that apart from LK-1 (panel A), heat shock treatment does not change expression of HLA-DR (B) or CD14 (C), nor does it lead to binding of irrelevant MAbs (D).

the clinical observation that viral infections that cause suppression of cellular immunity, for example, measles (22-24) or cytomegalovirus infection (W. Kuis, personal communication)), may lead to remission of disease activity in patients with JCA. Furthermore, chronic arthritis is found in patients with B-cell deficiencies such as common variable immunodeficiency, X-linked agammaglobulinemia and selective immunoglobulin A (IgA) deficiency (25,26). The clinical features of the chronic arthritis in patients with B-cell defects are indistinguishable from the disease in (B-cell competent) patients with JCA. Because the etiology of JCA has not been established and may even be variable, it can not be excluded that the underlying cause for the chronic arthritis in both patient groups differs. In patients with primary T-cell deficiencies, chronic arthritis is not observed.

Evidence for the fact that endogenous hsp 60 may act as an autoantigen in arthritis in humans was provided by the observation that T cells from synovial fluid and peripheral blood of patients with JCA can be activated when cultured in vitro with recombinant human hsp 60 (27). In our hands, adult patients with RA, healthy children, nor healthy young adults show a T-lymphocyte proliferative response to human hsp 60 (27). This is in accordance with a recent study of Pope et al. (28), who found no positive response to human hsp 60 of peripheral blood mononuclear cells from 11 adult patients with RA and 8 patients with other forms of inflammatory synovitis. However, synovial fluid mononuclear cells from 3 of 11 RA patients did respond to human hsp 60 (28). Activation of peripheral blood lymphocytes of normal healthy individuals with killed Mycobacterium tuberculosis results in cytotoxic T-cell activity for autologous target cells primed with synthetic peptides containing homologous sequences of mycobacterial and human hsp 60 (29). This indicates that T cells with a specificity for epitopes of conserved sequences in hsp 60 are present in peripheral circulation of normal individuals. Apparently, owing to regulatory control exerted by suppressor T cells or otherwise, these T cells do not become activated under physiological conditions.

When peripheral blood T-cells of patients with JCA were cultured with either human hsp 60 or mycobacterial hsp 60, we found a statistically significant correlation between the stimulation indices (r = 0.948, p < 0.02; [27]), suggesting that the T-cell response to human hsp 60 is at least in part directed against conserved sequences within the hsp 60 molecule. In a number of patients, a proliferative T-cell response could also be induced by activation with mycobacterial hsp 70 or the mycobacterial hsp 60 nonapeptide 180-188. Whether the positive proliferative response to mycobacterial hsp 70 is due to recognition of conserved sequences present in the human hsp 70 as well is sofar unknown. We can only conclude that T lymphocytes of patients JCA recognize next to mycobacterial hsps autologous hsps also. Based on these results, it has become clear that an endogenous hsp can serve as an autoantigen and lead to substantial T-lymphocyte reactivity in patients with JCA.

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